a novel n-cor complex contains components of the mammalian
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A novel N-CoR complex contains components of the mammalian SWI/SNF complex and
the corepressor KAP-1.
Caroline Underhill1, Majdi S.Qutob3, Siu-Pok Yee 2,3, Joseph Torchia1,2*
Cancer Research Laboratories, London Regional Cancer Centre, Dept. of Pharmacology and Toxicology1 and Oncology2 and Biochemistry 3 The University of
Western Ontario, London, Ontario, Canada.
*Corresponding Author: Dr. Joseph Torchia Cancer Research Laboratories. Rm 4020 London Regional Cancer Centre 790 Commissioners Road East London, Ontario Canada, N6A 4L6 Fax: (519) 685-8646 Tel: (519) 685-8692 E-mail: jtorchia@julian.uwo.ca
Running Title: Purification of Corepressor Complexes.
Key Words: repression, nuclear hormone receptors, deacetylase, purification.
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 29, 2000 as Manuscript M007864200 by guest on M
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Summary
Transcriptional silencing by many transcription factors is mediated by the Nuclear
Receptor Corepressor (N-CoR). The mechanism by which N-CoR represses basal
transcription involves the direct or indirect recruitment of histone deacetylases (HDACs).
We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2,
which possess histone deacetylase activity that is mediated by distinct HDACs. Based on
western blotting using antibodies against known subunits, the only HDAC found in the N-
CoR-1 complex is HDAC3. In contrast, N-CoR-2 contains predominantly HDAC1 and
HDAC2, as well as several other subunits that are found in the Sin 3A/HDAC complex.
Using mass spectrometry and western blotting, we have identified several novel
components of the N-CoR-1 complex which includes the SWI/SNF-related proteins BRG1,
BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing
at heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-
CoR co-localize throughout the nucleus. These results suggest that N-CoR is found in
distinct multiprotein complexes, which are involved in multiple pathways of transcriptional
repression.
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Introduction
Cellular proliferation and differentiation is critically dependent on the ability of sequence
specific DNA-binding transcription factors to activate or repress the transcription of target genes
in a coordinated fashion. This is accomplished through transcription factor-mediated recruitment
of coactivators and corepressors which can regulate transcription through the modification of the
local chromatin environment and by specifically interacting with components of the core
transcriptional machinery (1,2). The nuclear hormone receptor superfamily provides a unique
model to study the mechanism of transcriptional activation and repression and the role of
reversible chromatin modification in the control of gene expression. In the absence of ligand,
nuclear hormone receptors such as the thyroid hormone (T3R) and retinoic acid receptors (RAR)
function as potent repressors by interacting with specific corepressor proteins (3,4). Ligand
binding induces a conformational change in these receptors that results in corepressor release and
the recruitment of coactivator proteins (2,5). The Nuclear Receptor Corepressor (N-CoR), and its
related family member Silencing Mediator for Retinoid and Thyroid hormone receptor (SMRT),
were initially identified by yeast two hybrid screening using unliganded T3R or RAR as bait,
respectively (3,4). Several lines of evidence suggest that both N-CoR and SMRT mediate the
repressive effects of nuclear hormone receptors. First, both N-CoR and SMRT contain two
nuclear receptor interaction domains (IDs) in the carboxy terminus. Molecular characterization
of the ID’s reveals the presence of a signature CoR box motif that is necessary and sufficient for
receptor interaction and ligand-induced release of N-CoR or SMRT (6-8). More recently, a
strong correlation between repression mediated by the T3R and recruitment of N-CoR or SMRT
have also been demonstrated in Xenopus oocytes (9,10). Second, microinjection of anti-N-CoR
antibodies into living cells blocks T3R- and RAR-mediated repression (11).
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N-CoR and SMRT contain three highly conserved repression domains designated RD1,
RD2 and RD3 (3,4,12-14). The lack of homology between these domains suggests that their
mechanism of action is mediated via distinct pathways. RD1 serves as a major interacting
surface for the corepressor Sin3A, which, in turn, can directly interact with the histone
deacetylase 1 and 2 (HDAC1 and 2), suggesting that repression by N-CoR and SMRT is linked
to the deacetylation of histones (11,14,15). Single cell microinjection studies using antibodies
against mSin3, or its associated histone deacetylases, inhibits repression by unliganded T3R and
RAR (11). Transcriptional repression by nuclear hormone receptors is also blocked by
deacetylase inhibitors such as Trichostatin A (TSA) (11). Taken together, these studies suggest
that the repressive effects of N-CoR and SMRT are mediated, in part, by a Sin3/HDAC complex.
In contrast, the RD3 of SMRT and N-CoR interacts with the class II histone deacetylases that are
structurally related to the yeast Hda1 protein (16,17). The interaction between SMRT and class II
HDACs occurs in vivo and in vitro and correlates with the repressor activity of RD3.
Recent studies have also implicated N-CoR and SMRT in transcriptional repression that
is mediated by several other families of transcription factors including Notch (18), the
homeodomain proteins RPX and Pit-1 (19), p53 (20) and the antagonist-bound estrogen and
progesterone receptors (21, 22). Abnormal recruitment of N-CoR and SMRT has also been
linked to pathological disorders such as acute promyelocytic leukemia (PML) that is associated
with rearrangements of the gene encoding the RARα receptor and the PML zinc finger protein.
This results in the formation of a PML-RAR fusion protein that blocks myeloid differentiation.
Treatment with retinoic acid causes the release of N-CoR/SMRT and abolishes the RAR-PML-
mediated differentiation block (23, 24).
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Biochemical purification of the mammalian Sin3 has identified a Sin3 complex which
consists of 8-10 proteins and includes HDAC1 and 2, RbAp46/48, Sap 18 and Sap 30 (25, 26).
However, neither N-CoR nor SMRT were identified as components of the mSin3/HDAC
complex, or a related complex known as NURD/Mi2/NRD, which contains both histone
deacetylase and nucleosome remodelling activity (27-29). Based on these studies, N-CoR may
not be a stable component of a deacetylase complex and may function as an adaptor protein for
sequence-specific transcriptional repressors upon activation of specific signalling pathways.
Alternatively, it has been suggested that N-CoR may be targeting a different subpopulation of
cellular mSin3 or HDACs (30).
To address this issue, we have undertaken a biochemical purification of endogenous N-
CoR from HeLa cell nuclear extracts by combining conventional chromatography with
immunoaffinity purification using an affinity purified anti-N-CoR antibody. We have isolated
two major N-CoR-containing complexes designated N-CoR-1 and N-CoR-2 which, on the basis
of gel filtration chromatography, are each approximately 2.0 MDa. Both complexes possess
intrinsic histone deacetylase activity based on their ability to deacetylate core histones. We
demonstrate that N-CoR-2 contains proteins that are found in the Sin3/HDAC and
NURD/Mi2/NRD complexes, which are not found in N-CoR-1 suggesting that N-CoR-1 is a
novel N-CoR-containing complex. Using mass spectrometry we have identified several
components of the N-CoR-1 complex that includes several subunits of the SWI/SNF-related
chromatin remodelling complexes as well as the corepressor protein KAP-1. Taken together,
these findings implicate N-CoR in multiple pathways of transcriptional repression that may
involve the recruitment of distinct corepressor complexes.
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Experimental Procedures
Western blotting and antibody production
Subcellular fractions of cells were prepared according to standard methods (31). Western
blotting was performed as described previously (32). Protein samples were separated by SDS-
PAGE, transferred to nitrocellulose and detected by Enhanced Chemiluminescence according to
the manufacturers recommendations (Amersham). The HDAC2 antibody and the Sin3A antibody
were purchased from Santa Cruz Biotechnology. The RbAp48 antibody was from Upstate
Biotechnology. The N-CoR polyclonal antisera was raised against a His-tagged recombinant N-
CoR (aa 2232-2453) protein and was purified by protein A Sepharose chromatography. To
further purify the anti-N-CoR antibody, the IgG fraction was passed through an affinity column
consisting of the His-tagged recombinant N-CoR protein (aa 2232-2453) crosslinked to
Sepharose 4B, and the specific antibody was eluted with 100 mM glycine (pH 2.8).
Purification of the N-CoR complexes
Forty litres of HeLa cells, grown to mid-log phase, were obtained from the National Cell Culture
Centre (Minneapolis, MN). Nuclear extracts were prepared according to standard methods (31).
To purify the nuclear N-CoR complexes, the nuclear extract was dialyzed against buffer A (20
mM Tris-HCl, pH 7.9, 0.5 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.2 mM
PMSF and 5 µg/ml each of leupeptin, aprotinin and pepstatin A) containing 100 mM KCl and
was loaded onto an 80 ml P11 phosphocellulose column pre-equilibrated with the same buffer.
The flowthrough was collected and the column was washed sequentially in a stepwise fashion
using buffer A containing 0.3 M, 0.5 M and 1.0 M KCl. At each step, the column was washed
with two column volumes of buffer containing the corresponding salt concentration to allow UV
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absorbance to stabilize near baseline prior to collection of the next protein peak. The majority of
immunoreative N-CoR was found in the 0.3 and 0.5 M KCl fractions. The N-CoR-containing
fractions were pooled, dialyzed against buffer A containing 100 mM KCl and then passed
through a DEAE Sepharose column and eluted with an increasing KCl gradient. All fractions
were analyzed by western blotting using anti-N-CoR antibody, the N-CoR-containing fractions
were again pooled, concentrated by precipitating with 20-60% ammonium sulphate and then
applied to a Sephacryl S300 column pre-equilibrated with buffer A containing 100 mM KCl.
The column was washed with buffer A at a flow rate of 0.4 ml/min. Each N-CoR complex eluted
as a single large molecular mass peak of approximately 2.0 MDa. The fractions corresponding to
each peak were analyzed for N-CoR by western blotting, pooled, and dialyzed against buffer A
containing 100 mM KCl without DTT. For affinity purification of the N-CoR complexes,
affinity purified N-CoR antibody was crosslinked to protein A Sepharose using
dimethylpalmilidate (DMP) according to standard procedures (33). The pooled fractions from the
gel filtration step were precleared using protein A Sepharose crosslinked to rabbit IgG. The
precleared sample was then loaded onto the affinity column at a flow rate of 0.4 ml/min and the
flowthrough was collected and reloaded onto the affinity column five times. The bound proteins
were washed with ten column volumes each of buffer A (without DTT) containing 0.1% NP40
and 0.3M KCl, 0.1% NP40 and 0.5M KCl, 0.5% NP40 and 0.7M KCl, and a final wash in buffer
A containing 100 mM KCl prior to elution with 100 mM glycine (pH 2.8). For mock purification
experiments, samples from the gel filtration step were loaded onto protein A sepharose alone, or
protein A sepharose crosslinked to an irrelevant antibody.
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Mass spectrometry
Proteins were separated by 7% SDS-PAGE and then stained with colloidal blue for one hour
followed by destaining in 25% MeOH for an additional 2 hrs. The protein bands were excised
and cut into 1 mm3 pieces. The gel pieces were washed twice in a 50% CH3CN solution for 5
min followed by two washes with a 250 µl solution consisting of 50% CH3CN/50 mM
NH4HCO3 for 30 min. The gel pieces were lyophilized, rehydrated in 10 mM NH4HCO3 (pH 8.5)
containing 0.1 µg/µl trypsin (Roche) and incubated overnight at 37EC. The tryptic fragments
were extracted by two 30 min washes with a solution containing 60% CH3CN/10% TFA. The
combined solutions were lyophilized using a speedvac, resuspended in 20 µl 0.5% TFA solution
and the peptide suspensions were purified using a ZipTip (Millipore) cartridge according to the
manufacturers instructions prior to analysis by MALDI-MS.
MALDI-MS analyses were carried out by using a Perseptive Biosystem Voyager-DE
STR mass spectrometer (Perseptive Biosystems Inc., Farmingham, MA), equipped with a pulsed
UV nitrogen laser (337 nm, 3-ns pulse) and a dual microchannel plate detector. The spectra were
acquired in reflectron-DE mode, acceleration voltage was set to 20 kV, grid voltage at 72% of
the acceleration voltage, guide wire voltage at 0.020%, delay time at 140 ns, low mass gate was
set at 250 Da, and the mass to charge ratio was calibrated internally with the dimer of α-cyano-4-
hydroxycinnamic acid ([2M+H]+ 380.09 Da) and trypsin autolysis peptide ([M+H]+ 2163.05 Da).
Two parts of α-cyano-4-hydroxycinnamic acid and one part of nitrocellulose were dissolved in
acetone-isopropanole (4:1) to final concentrations of 20 and 10 mg/ml, respectively. A 0.5 µl
volume of this solution was deposited on MALDI target and allowed to dry. 0.5 µl of 1% acetic
acid was placed on top of the matrix layer followed by addition of 1 µl of analyte solution. Mass
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spectra were recorded after evaporation of the solvent and processed using GRAMS software for
data collection and analysis.
Peptide sequence analysis was conducted by Post-Source Decay (PSD) technique. The
spectra were acquired at DE-reflectron mode. The accelerating voltage was set at 20 KV, grid
voltage at 75%, guide wire voltage at 0.024%, and delay time at 100ns. The timed ion selector
was pre-set to the protonated molecular weight of the analyte. The spectra were acquired in 10-
13 segments with mirror ratio 1.0 to 0.1342 and finally "stitched" together by the instrument
software. The tryptic peptides obtained were used to search for protein candidates in the
nonredundant protein sequence database with the program PROWL that is publicly available
(http://prowl.rockefeller.edu).
Immunofluorescence
HeLa cells were plated onto Fisherbrand microscope slides and maintained in DMEM
with 10% FBS at 37°C with 5% CO2 . Cells were allowed to adhere for approximately 18 hr
prior to fixation with 4% paraformaldehyde for 10 min. The cells were then permeabilized with
0.2% Triton X-100 in PBS and blocked for 30 min in 5% goat serum. After a brief wash, slides
were incubated with rabbit anti-N-CoR antibody (1:300 dilution) and anti-KAP-1 mouse
monoclonal IgG antibody supernatant (1:10 dilution) in 3% BSA at 4EC overnight. Cells were
then washed three times in PBS for 5 min, and then incubated for 4 hrs at room temperature with
anti-mouse IgG conjugated to rhodamine (1:500 dilution) and anti-rabbit IgG conjugated to
fluorescenin (1:500 dilution) (Santa Cruz). Images were captured using a Sensicam digital
camera on a Olympus Provis AX-70 microscope and analyzed using Image Pro Plus version 4.0.
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Purification of radiolabelled HeLa core histones
Core histones were purified from HeLa cells essentially as described with some
modifications (34). Briefly, 1 liter of HeLa cells were grown to a density of 0.8x106 cells/ml in
DMEM supplemented with 7% FBS. The cells were preincubated with 10 mM sodium butyrate
for 24 hrs before harvesting. The cells were then centrifuged and resuspended in 50 ml of PBS
supplemented with 100 µg/ml cyclohexamide, 10 mM sodium butyrate and 0.1 mCi/ml [3H]
acetic acid and incubated at 37EC for 2hrs with gentle stirring. The cells were then washed three
times with PBS (500 x g, 5 min) and then lysed in 10 ml of buffer B (20 mM Tris-HCl (pH 7.6) ,
2 mM MgCl2, 3mM CaCl2, 10 mM sodium butyrate, 0.2 mM PMSF and 1% NP40). Nuclei were
collected by centrifugation (2,000xg for 10 min), washed twice in 10 ml of buffer B and then
resuspended in buffer B without NP40 (buffer C). The nuclear proteins were extracted by adding
buffer C containing 0.42 M KCl, followed by centrifugation at 10,000xg for 30 min. The nuclear
pellet was resuspended in two volumes of buffer C and then extracted twice with 0.2 M H2S04
for 90 min in an icebath with gentle stirring then centrifuged (30,000xg for 20 min). The
supernatants were pooled, dialyzed extensively against 100 mM acetic acid and the histones were
precipitated with ten volumes of ice-cold acetone and resuspended in water to a concentration of
approximately 2 mg/ml.
Histone deacetylase activity
Histone deacetylase activity was monitored as described (35). Aliquots of the affinity purified
complexes were retained on 25µl of Protein A Sepharose affinity resin containing crosslinked
anti-N-CoR antibody. The beads were washed extensively with buffer A containing 500 mM
KCl and 0.5% NP40 and then resuspended in buffer D (20 mM Tris-HCl (pH 7.6) 100 mM KCl,
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0.1 mM EDTA, 0. 2 mM DTT and 0.2 mM PMSF) containing [3H]histones (20,000 cpm/rxn) to
a final volume of 200 µl. The reactions were allowed to proceed at 37EC for 90 min and stopped
by the addition of 0.05 ml of 0.1 M HCl/0.7 M acetic acid solution. Released [3H]acetate was
extracted with 0.6 ml ethyl acetate. After centrifugation, 0.3 ml of the upper organic phase was
used for liquid scintillation counting.
Results
Identification of N-CoR-containing complexes.
As a first step in determining whether endogenous N-CoR is a stable component of a corepressor
complex, we generated a polyclonal antibody against the carboxy terminus of N-CoR. The
affinity purified antibody recognized a single protein band of approximately 260 kDa that is
consistent with the size of N-CoR (Fig 2A). Nuclear extracts prepared from HeLa cells were
initially fractionated on a P11 phosphocellulose column using a step gradient with increasing salt
concentrations (Fig.1A and 1B). Western blot analysis of the eluates derived from this column
demonstrated that N-CoR was present in three fractions. A distinct N-CoR-containing fraction
was eluted with 0.3 M KCl buffer (N-CoR-1). N-CoR immunoreactivity was also found in
fractions eluted with 0.5 M (N-CoR-2) and a minor component was also detected in the 1.0 M
KCl (data not shown). To confirm that efficient protein separation was achieved with the P11
column, we analyzed the eluates for the transcriptional coactivator p/CIP (32), which is part of a
family of nuclear receptor coactivator proteins required for ligand dependent signalling. p/CIP
eluted from the phosphocellulose column with 0.1 M KCl. A similar elution profile was obtained
with other members of this family of coactivators (data not shown) suggesting that they are
found in complexes that are distinct from N-CoR.
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The relative abundance of Sin3A was also examined in the fractions eluted from the
phosphocellulose column. Although Sin3A was detected in all of the N-CoR-containing
fractions, the relative distribution was not consistent with the distribution of N-CoR. Based on
densitometric scanning, only 7% of the total amount of Sin3A was found in the 0.3 M KCl
fraction suggesting that the major N-CoR-containing fraction is not complexed to Sin3A but may
function in other contexts (16,17).
To characterize these complexes further, we subjected the P11-purified fractions to
purification by DEAE-Sepharose followed by gel filtration chromatography using a Sephacryl S-
300 column. Immunoblot analysis of the eluates derived from the Sephacryl S300 column
demonstrated that N-CoR-1 and N-CoR-2 eluted with the void volume corresponding to a
molecular mass of approximately 2.0 MDa (Fig. 1C). Since N-CoR is a 260 kDa protein, this
result strongly suggests that it is found as part of large multiprotein complexes. To purify these
complexes further, the affinity purified N-CoR antibody was crosslinked to protein A Sepharose
and the N-CoR-containing fractions isolated from the Sephacryl S300 column were pooled and
applied to the immunoaffinity column. After extensive washing with highly stringent buffer
containing 0.5% NP40 and 0.7M KCl, the retained proteins were eluted with 100 mM glycine
(pH2.8).
Western blot analysis of the immunopurified N-CoR-1 complex demonstrated that the
majority of N-CoR bound to the immunoaffinity column and that a single immunoreactive peak
was present in the eluate with no significant breakdown products detected (Fig. 2A). Silver
staining of an SDS-PAGE gel of the N-CoR-1 complex consistently identified approximately 20
proteins ranging in molecular weight from 35 to 350 kDa (Fig. 2B). The interaction of these
polypeptides appears to be specific since the majority of these are not retained when an
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analogous “mock” purification was performed using a control affinity column. Since the
mechanism of N-CoR action has been linked to the recruitment of a Sin3A/HDAC complex, we
initially tested the eluate by western blotting using antibodies against specific subunits found in
this complex (Fig. 2C). Although Sin3A, HDAC1 and 2 migrated through the Sephacryl S300
column as large molecular weight complexes and appeared to coelute with N-CoR (data not
shown), they were not retained by the anti-N-CoR immunoaffinity column. This suggests that
they are not intrinsic components of the N-CoR-1 complex and are most likely associated with
other multiprotein complexes. The only component that was detected by western blotting was
HDAC3, which is consistent with recent observations (9, 10, 36, 37). In addition, we were
unable to identify either RbAp48, which has been demonstrated to be a common subunit found in
several chromatin modifying complexes, or Sap 30 a component of the Sin3/HDAC complex.
Taken together, these observations suggest that the N-CoR-1 complex contains novel
components.
The finding that HDAC3 is a component of the N-CoR-1 complex prompted us to
examine whether N-CoR-1 complex also possesses deacetylase activity. [3H]-labelled core
histones were purified from HeLa cells, preincubated with immunopurified N-CoR-1 and the
deacetylase activity was monitored by quantifying the release of radiolabelled acetyl CoA. We
observed that N-CoR-1 can deacetylate core histones in this assay, consistent with the presence
of HDAC3, and that the deacetylation activity is blocked when the complex was preincubated
with the deacetylase inhibitor TSA (Fig. 2D).
Mass spectrometric analysis of the N-CoR-1 complex.
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To identify novel components of the N-CoR-1 complex, SDS-PAGE analysis was performed and
several of the protein bands were excised and subjected to in-gel tryptic digestion. The peptides
were then isolated and analyzed by mass spectrometry (Table 1, Fig. 3). The majority of the
tryptic fragments derived from the 300 kDa protein band corresponded to N-CoR. We also
detected several tryptic fragments corresponding to the related family member SMRT although
these were relatively minor components Interestingly, several of the proteins which consistently
copurified with N-CoR have been found as intrinsic components of unrelated, yet biologically
important complexes. For example, SAP 130 and SF3a120 are subunits of protein complexes
essential for spliceosome assembly (38, 39). However, a function for SAP 130 and SF3a120 in
transcriptional regulation has not been previously uncovered.
Four of the proteins identified by mass spectrometry belong to the SWI/SNF family of
proteins and include the BRG1-Associated Factor (BAF) 170, BAF 155, BAF 47/INI1 and the
SWI/SNF Related CBP Activator Protein (SRCAP) (40). BAF 170 and BAF 155 are highly
similar homologues of the yeast SWI3 protein whereas BAF 47/INI1 represents the mammalian
homologue of the yeast SNF5 protein. All three proteins have been identified as subunits of
several SWI/SNF-related chromatin remodeling complexes including the human BRG1 and
hBRM complexes (41,42). We were unable to obtain definitive evidence for the BRG1
component of the SWI/SNF complex using mass spectrometry. However western blotting using
an anti-BRG1 antibody subsequently demonstrated that the p190 subunit is BRG1 suggesting
that the four core components of the SWI/SNF complex are found in the N-CoR-1 complex (Fig.
4).
To verify that these components co-localize with N-CoR, western blotting was performed
using polyclonal antibodies that recognize either BRG1, BAF 170, BAF 155 or BAF 47. We
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found that all of these proteins eluted as a high molecular weight complex from the Sephacryl
S300 column. Although the overlap with N-CoR was clearly significant, it was not identical
suggesting that the association of N-CoR with SWI/SNF is not 100%. (Fig. 4A). Importantly, all
of these same proteins are found in the immunoaffinity purified N-CoR-1 complex (Fig. 4B),
consistent with the mass spectrometry data.
KAP-1 and N-CoR colocalize in vivo.
The 110 kDa protein identified by mass spectrometry corresponds to Krab-associated
protein 1 (KAP-1) also known as TIF1β (43,44). KAP-1 is a 97 kDa transcriptional corepressor
that interacts with proteins containing the Kruppel associated box domain (KRAB), a common
motif found in many DNA binding transcriptional repressor proteins. Recent observations have
also demonstrated that KAP-1 interacts in vivo and in vitro with members of the heterochromatin
protein 1 (HP-1) family, which are important for regulating heterochromatin-mediated gene
silencing (45,46). HP-1 proteins have also been implicated in position effect variegation, a
euchromatic silencing mechanism exhibited by genes placed within or adjacent to
heterochromatin (47). Interestingly, it has been shown that TSA can interfere with KAP-1/TIF1β
mediated repression (48). This suggests that the mechanism of repression mediated by KAP-1
also involves histone deacetylation and is consistent with the presence of HDAC3 in the N-CoR-
1 complex.
To verify that KAP-1 co-localizes with N-CoR, western blotting was performed using
polyclonal antibodies which recognize either N-CoR or KAP-1. We found that a significant
component of KAP-1 also copurified with N-CoR as a high molecular weight complex from the
Sephacryl S300 column (Fig. 5A), and was also present in the immunoaffinity purified N-CoR-1
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complex (Fig. 5B). To define a potential in vivo role for KAP-1/N-CoR association, we
performed indirect immunoflourescence in asynchronously growing HeLa cells using a
monoclonal antibody raised to KAP-1( kindly provided by Dr. F. Rauscher III) together with
affinity purified anti-N-CoR antibody. Both KAP-1 and N-CoR staining were found throughout
the nucleus as an even speckled pattern (Fig. 5C). In some regions, both N-CoR and KAP-1 were
concentrated in micropunctate-like structures which may represent regions of pericentromeric
heterochromatin. When KAP-1 and N-CoR staining were directly compared, a significant
component of the signals overlap particularly in the granular regions presumed to be
pericentromeric heterochromatin. Taken together, these data indicate that a major fraction of
KAP-1 and N-CoR are found in the same complex in vivo and may function through a similar
mechanism.
Identification of the N-CoR/Sin3/HDAC complex
A similar purification scheme was used to affinity purify the N-CoR-2 complex.
However, SDS-PAGE analysis revealed a different protein profile when compared to the N-
CoR-1 complex (Fig. 6A). In this case, approximately 12-15 proteins consistently copurified with
N-CoR. Surprisingly, we observed that several of the proteins found in the Sin3/HDAC complex
are also found in the immunopurified N-CoR-2 complex including Sin3A, HDAC1 and HDAC2
and Sap30 (Fig. 6B). HDAC3 was detected in the N-CoR-2 complex although it does not appear
to be a stoicheometric component. We could not detect RbAp48 although it was clearly present
in the Sephacryl S300 fraction.
To assess whether N-CoR-2 also possesses histone deacetylase activity, [3H]-labelled
core histones purified from HeLa cells were preincubated with the immunopurified complexes
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and the deacetylase activity was monitored by quantifying the release of radiolabelled acetyl
groups. N-CoR-2 deacetylated core histones in this assay, consistent with the presence of
HDAC1 and HDAC2, and the deacetylase activity is blocked when the complex was
preincubated with the deacetylase inhibitor TSA (Fig. 6C). These data provide direct
biochemical evidence for the existence of an N-CoR/Sin3/HDAC complex, consistent with
earlier reports (11,14).
Discussion
Functional studies using microinjection of neutralizing antibodies into living cells have
provided strong evidence suggesting that the repressive effects of N-CoR and SMRT are
mediated, in part, through the recruitment of a Sin3/HDAC complex (11,14). However,
biochemical purification studies have been unable to demonstrate the existence of an endogenous
N-CoR/Sin3A/HDAC complex (17,25,26). Two hypotheses have been proposed to explain these
results. First, it is possible that the interactions between N-CoR or SMRT and the Sin3/HDAC
complex are transient, or require the recruitment of additional factors to become stable
components (49). Second, that multiple N-CoR complexes exist which contain distinct subunits
and become associated with different classes of transcription factors. Partial evidence for this
hypothesis has come from studies using confocal microscopy which demonstrated that the
distribution of N-CoR is heterogenous and that only a small fraction of endogenous N-CoR is co-
localized with HDAC1(30).
In the present study, we have used phosphocellulose chromatography to separate
endogenous N-CoR into two chromatographically distinct fractions designated N-CoR-1 and N-
CoR-2. We have purified N-CoR-1 and N-CoR-2 to apparent homogeneity and have used both
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western blotting and mass spectrometry to demonstrate that each complex contains distinct
proteins. For example, although N-CoR-1 and N-CoR-2 both possess HDAC activity, this
activity is mediated by different HDACs. HDAC3 represents the major class I deacetylase in the
N-CoR-1 complex, whereas HDAC1 and 2 are found only in the N-CoR-2 complex. The
presence of different catalytic subunits in each of the N-CoR complexes suggests that they may
have unique functional roles in the cell.
Our results are consistent with recent findings demonstrating that HDAC3 is part of a
complex, which also includes N-CoR or SMRT (9,10,36,37). It is interesting that the number of
proteins which we have identified in the N-CoR complexes is considerably greater than the
number of proteins identified in the SMRT core complex. This suggests that there are intrinsic
differences between SMRT and N-CoRs ability to interact with other intracellular proteins.
In addition to HDAC3, the N-CoR-1 complex also contains several proteins found in the
SWI/SNF complex (50). The SWI/SNF complex is the prototypical chromatin remodelling
complex which has been shown to disrupt chromatin structure and facilitate the binding of
transcriptional activators to nucleosomal sites (51-53). The glucocorticoid receptor (GR) can
target the SWI/SNF complex to chromatinized templates containing GR binding sites in yeast
and in mammalian cells resulting in disruption of local nucleosomal structure (54, 55). Multiple
mammalian SWI/SNF complexes have also been identified which have diverse subunit
compositions although BRG1, BAF 170, BAF 155 and BAF 47 represent the core catalytic
components (41,42). BRG1 is homologous to the yeast SWI2/SNF2 and possesses DNA-
dependent ATPase activity, a necessary function carried out by all chromatin remodelling
complexes identified to date. Reconstitution studies have shown that BRG1 alone can remodel
nucleosomes, although this remodeling activity is enhanced when all four core components are
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present (56). Thus, one possibility is that the core BAFs may be involved in targeting or
stabilizing nucleosomes in a particular conformation that is conducive to remodelling activity by
BRG1.
The presence of BRG1 strongly suggests that the N-CoR-1 complex possesses chromatin
remodeling activity. This hypothesis is consistent with recent studies demonstrating that
chromatin remodeling is required for transcriptional repression as well as activation. Genome-
wide expression analysis using DNA microarrays have shown that mRNA levels for many genes
are elevated in yeast mutants carrying mutations for SWI/SNF (57,58). In mammalian cells, Rb
forms a complex with hSWI/SNF to inhibit transcription of cyclin E and A, resulting in growth
arrest (59,60). Finally, several labs have identified multiprotein chromatin remodeling complexes
which are believed to be directly involved in transcriptional repression. One such complex,
known as NuRD or Mi2, possesses both histone deacetylase and nucleosome remodeling activity
suggesting that chromatin modifying enzymes can be coupled to regulate transcription in vivo
(27-29). Thus, chromatin remodelling may be necessary step to facilitate the binding of N-CoR
by specific factors associated with nucleosomal DNA. Interestingly, the gene encoding BAF
47/INI 1 has been found mutated or deleted in various rhabdoid sarcoma tumour cell lines and in
primary rhabdomyosarcoma (61). This suggests that the repressive effects mediated by the N-
CoR-1 complex represents a critical mechanism for the regulation of specific genes important for
muscle cell proliferation
We have also established that KAP-1 is an intrinsic component of the N-CoR-1 complex.
KAP-1 functions as a bona fide corepressor involved in mediating the repression of a large
family of Kruppel-like zinc finger proteins which contain the KRAB repression domain (KRAB-
ZFP’s) (41,42). KAP-1 binds to multiple KRAB-containing zinc finger proteins in vivo and in
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vitro and mutations that abolish repression also abolish interaction with the KRAB domain. A
mechanistic link between KAP-1 and histone modification has also been established with the
demonstration that TSA can interfere with KAP-1-mediated repression (46). It has recently been
demonstrated that KAP-1 associates with members of the heterochromatin protein 1 (HP1)
family in vivo and in vitro (43,45). HP1 proteins are conserved nonhistone chromosomal proteins
associated primarily with pericentric heterochromatin where it is believed they function as
regulators of hetrochromatin silencing. The finding that KAP-1 and N-CoR colocalize in vitro
and in vivo suggests that their mechanism of action is similar. Importantly, N-CoR, through
KAP-1-directed recruitment of HP-1 proteins, may be involved in the assembly and/or
maintenance of heterochromatin at specific sites within the genome suggesting an entirely novel
function for N-CoR.
Based on western blotting using selected antibodies, N-CoR-2 contains several proteins
found in the Sin3A/HDAC complex. These results are in contrast to studies which have failed to
demonstrate the presence of such a complex (9, 10, 36). There may be several possible
explanations for these differences. First, purification strategies, which rely on the use of specific
antibodies, could disrupt specific protein-protein interactions. Second, the presence of a pre-
existing complex may be cell cycle dependent, or its composition can be regulated through post-
translational modifications. Consequently, variations in growth conditions might determine the
relative abundance of a specific multisubunit complex. Finally, variability in the composition of
the initial starting material may be important for determining the existence of specific
complexes.
In conclusion, we have provided biochemical evidence for the existence of multiple
endogenous N-CoR complexes. The finding that these complexes possess distinct factors
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suggests that they may perform gene-specific functions by utilizing multiple mechanisms of
transcriptional regulation. The continued analysis of the megadalton N-CoR complexes is critical
to deciphering common and unique mechanisms of gene repression that are necessary for the
homeostatic control of differentiated cell function.
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References
1. Collingwood, T. N., Urnov, F. D., and Wolffe, A. P. (1999) J Mol Endocrinol 23,
255-75
2. Glass, C. K., and Rosenfeld, M. G. (2000) Genes Dev 14, 121-41
3. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R.,
Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K., and et al. (1995) Nature 377,
397-404
4. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-7
5. Hu, I., and Lazar, M. A. (2000) Trends Endocrinol Metab 11, 6-10
6. Hu, X. and Lazar, M. A. (1999) Nature 402, 93-6 7. Perissi, V., Staszewski, L. M., McInerney, E. M., Kurokawa, R., Krones, A.,
Rose, D. W., Lambert, M. H., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G.
(1999) Genes Dev. 13, 3198-208
8. Nagy, L., Kao, H. Y., Love, J. D., Li, C., Banayo, E., Gooch, J. T., Krishna, V.,
Chatterjee, K., Evans, R. M., and Schwabe, J. W. Genes Dev. (1999) 13: 3209-16
9. Urnov, F. D., Yee, J., Sach, L., Collingwood, T. N., Bauer, A., Beug, H., Shi, Y.,
and A.P., W. (2000) EMBO J 19, 4074-4090
10. Lee, J., Wang, J., Wang, J., Nawaz, Z., Liu, J. M., Qin, J., and Wong, J. (2000)
EMBO J 19, 1-10
11. Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D.,
Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman,
R. N., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 43-8
by guest on March 23, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Purification of corepressor complexes
23
12. Perissi, V., Staszewski, L. M., McInerney, E. M., Kurokawa, R., Krones, A.,
Rose, D. W., Lambert, M. H., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G.
(1999) Genes Dev 13, 3198-208
13. Nagy, L., Kao, H. Y., Love, J. D., Li, C., Banayo, E., Gooch, J. T., Krishna, V.,
Chatterjee, K., Evans, R. M., and Schwabe, J. W. (1999) Genes Dev 13, 3209-16
14. Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E.,
Schreiber, S. L., and Evans, R. M. (1997) Cell 89, 373-80
15. Hassig, C. A., Fleischer, T. C., Billin, A. N., Schreiber, S. L., and Ayer, D. E.
(1997) Cell 89, 341-7
16. Kao, H. Y., Downes, M., Ordentlich, P., and Evans, R. M. (2000) Genes Dev 14,
55-66
17. Huang, E. Y., Zhang, J., Miska, E. A., Guenther, M. G., Kouzarides, T., and
Lazar, M. A. (2000) Genes Dev 14, 45-54
18. Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M.,
Kintner, C. R., Evans, R. M., and Kadesch, T. (1998) Genes Dev 12, 2269-77
19. Xu, L., Lavinsky, R. M., Dasen, J. S., Flynn, S. E., McInerney, E. M., Mullen, T.
M., Heinzel, T., Szeto, D., Korzus, E., Kurokawa, R., Aggarwal, A. K., Rose, D.
W., Glass, C. K., and Rosenfeld, M. G. (1998) Nature 395, 301-6
20. Murphy, M., Ahn, J., Walker, K. K., Hoffman, W. H., Evans, R. M., Levine, A.
J., and George, D. L. (1999) Genes Dev 13, 2490-501
21. Smith, C. L., Nawaz, Z., and O'Malley, B. W. (1997) Mol Endocrinol 11, 657-66
22. Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T. M., Schiff, R.,
Del-Rio, A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C.
by guest on March 23, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Purification of corepressor complexes
24
K., Glass, C. K., Rosenfeld, M. G., and Rose, D. W. (1998) Proc Natl Acad Sci
USA 95, 2920-5
23. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H., Jr., and Evans, R. M.
(1998) Nature 391, 811-4
24. Grignani, F., De Matteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M.,
Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C., Lazar, M. A.,
Minucci, S., and Pelicci, P. G. (1998) Nature 391, 815-8
25. Zhang, Y., Iratni, R., Erdjument-Bromage, H., Tempst, P., and Reinberg, D.
(1997) Cell 89, 357-64
26. Zhang, Y., Sun, Z. W., Iratni, R., Erdjument-Bromage, H., Tempst, P., Hampsey,
M., and Reinberg, D. (1998) Mol Cell 1, 1021-31
27. Wade, P. A., Jones, P. L., Vermaak, D., and Wolffe, A. P. (1998) Curr Biol 8,
843-6
28. Xue, Y., Wong, J., Moreno, G. T., Young, M. K., Cote, J., and Wang, W. (1998)
Mol Cell 2, 851-61
29. Tong, J. K., Hassig, C. A., Schnitzler, G. R., Kingston, R. E., and Schreiber, S. L.
(1998) Nature 395, 917-21
30. Soderstrom, M., Vo, A., Heinzel, T., Lavinsky, R. M., Yang, W. M., Seto, E.,
Peterson, D. A., Rosenfeld, M. G., and Glass, C. K. (1997) Mol Endocrinol 11,
682-92
31. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res 11,
1475-89
by guest on March 23, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Purification of corepressor complexes
25
32. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and
Rosenfeld, M. G. (1997) Nature 387, 677-84
33. Harlow, E., and Lane, D. (1999) Using Antibodies: A laboratory manual. Cold
Spring Harbour Laboratory Press
34. Carmen, A. A., Rundlett, S. E., and Grunstein, M. (1996) J Biol Chem 271,
15837-44
35. Hendzel, M. J., Delcuve, G. P., and Davie, J. R. (1991) J Biol Chem 266, 21936-
42
36. Wen, Y. D., Perissi, V., Staszewski, L. M., Yang, W. M., Krones, A., Glass, C.
K., Rosenfeld, M. G., and Seto, E. (2000) Proc Natl Acad Sci U S A 97, 7202-7
37. Guenther, M. G., Lane, W. S., Fischle, W., Verdin, E., Lazar, M. A., and
Shiekhattar, R. (2000) Genes Dev 14, 1048-57
38. Kramer, A., Mulhauser, F., Wersig, C., Groning, K., and Bilbe, G. (1995) RNA 1,
260-72
39. Das, B. K., Xia, L., Palandjian, L., Gozani, O., Chyung, Y., and Reed, R. (1999)
Mol Cell Biol 19, 6796-802
40. Johnston, H. Kneer, J., Chackalaparampil, I. Yaciuk, P., and Chrivia, J. (1999) J.
Biol. Chem 274, 16370-16376
41. Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R., Muchardt,
C., Kalpana, G. V., Goff, S. P., Yaniv, M., Workman, J. L., and Crabtree, G. R.
(1996) EMBO J 15, 5370-82
42. Wang, W., Xue, Y., Zhou, S., Kuo, A., Cairns, B. R., and Crabtree, G. R. (1996)
Genes Dev 10, 2117-30
by guest on March 23, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Purification of corepressor complexes
26
43. Friedman, J. R., Fredericks, W. J., Jensen, D. E., Speicher, D. W., Huang, X. P.,
Neilson, E. G., and Rauscher, F. J., 3rd. (1996) Genes Dev 10, 2067-78
44. Moosmann, P., Georgiev, O., Le Douarin, B., Bourquin, J. P., and Schaffner, W.
(1996) Nucleic Acids Res 24, 4859-67
45. Ryan, R. F., Schultz, D. C., Ayyanathan, K., Singh, P. B., Friedman, J. R.,
Fredericks, W. J., and Rauscher, F. J., 3rd. (1999) Mol Cell Biol 19, 4366-78
46. Nielsen, A. L., Ortiz, J. A., You, J., Oulad-Abdelghani, M., Khechumian, R.,
Gansmuller, A., Chambon, P., and Losson, R. (1999) EMBO J 18, 6385-95
47. Wakimoto, B. T. (1998) Cell 93, 321-4
48. Le Douarin, B., Nielsen, A. L., Garnier, J. M., Ichinose, H., Jeanmougin, F.,
Losson, R., and Chambon, P. (1996) EMBO J 15, 6701-15
49. Laherty, C. D., Billin, A. N., Lavinsky, R. M., Yochum, G. S., Bush, A. C., Sun,
J. M., Mullen, T. M., Davie, J. R., Rose, D. W., Glass, C. K., Rosenfeld, M. G.,
Ayer, D. E., and Eisenman, R. N. (1998) Mol Cell 2, 33-42
50. Sudarsanam, P., and Winston, F. (2000) Trends Genet 16, 345-351
51. Peterson, C. L., and Herskowitz, I. (1992) Cell 68, 573-83
52. Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., and Green, M. R.
(1994) Nature 370, 477-81
53. Cote, J., Quinn, J., Workman, J. L., and Peterson, C. L. (1994) Science 265, 53-60
54. Fryer, C. J., and Archer, T. K. (1998) Nature 393, 88-91
55. Yoshinaga, S. K., Peterson, C. L., Herskowitz, I., and Yamamoto, K. R. (1992)
Science 258, 1598-604
56. Phelan, M. L., Sif, S., Narlikar, G. J., and Kingston, R. E. (1999) Mol Cell 3,
by guest on March 23, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Purification of corepressor complexes
27
247-53
57. Sudarsanam, P., Iyer, V. R., Brown, P. O., and Winston, F. (2000) Proc Natl Acad
Sci U S A 97, 3364-9
58. Sudarsanam, P., and Winston, F. (2000) Trends Genet 16, 345-351
59. Shanahan, F., Seghezzi, W., Parry, D., Mahony, D., and Lees, E. (1999) Mol Cell
Biol 19, 1460-9
60. Zhang, H. S., Gavin, M., Dahiya, A., Postigo, A. A., Ma, D., Luo, R. X., Harbour,
J. W., and Dean, D. C. (2000) Cell 101, 79-89
61. Versteege, I., Sevenet, N., Lange, J., Rousseau-Merck, M. F., Ambros, P.,
Handgretinger, R., Aurias, A., and Delattre, O. (1998) Nature 394, 203-6
62. Qian, Y. W., and Lee, E. Y. (1995) J Biol Chem 270, 25507-13
63. Qian, Y. W., Wang, Y. C., Hollingsworth, R. E., Jr., Jones, D., Ling, N., and Lee,
E. Y. (1993) Nature 364, 648-52
64. Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y. B., and Wolffe, A. P. (1999) Mol
Cell Biol 19, 5847-60
Acknowledgements
We are grateful to Dr. D. Ayer for supplying the Sap 30 antibody, to Dr. W.D. Wang for the
BAF 170, BAF 155 and BAF 47 antibodies, Dr. S. Schreiber for the HDAC-1 antibody and
special thanks to Dr. Rauscher III for the anti-KAP-1 monoclonal and polyclonal antibodies.
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Table Legends
Table 1. Proteins in the N-CoR-1 complex unambigously identified by mass spectrometry.
Figure Legends
Figure 1. Purification of N-CoR-containing complexes. (A) Schematic representation of the
various chromatographic steps used to purify the N-CoR complexes. (B) Western Blot analysis
of the fractions eluted from the P11 phosphocellulose column. The salt concentrations used to
purify the individual fractions are indicated at the top. For each sample, 20 µg of protein were
separated by SDS-PAGE, transferred to nitrocellulose and then probed with the specific
antibodies indicated on the left. (C) Gel filtration chromatography of the N-CoR complexes
using a Sephacryl S300 column. The molecular weight standards are indicated at the top of the
panel; 660 kDa, thyroglobulin; 440 kDa, ferritin.
Figure 2. N-CoR-1 is a multiprotein complex. (A) Western blot of the immunopurified N-CoR-1
complex. N-CoR-1 eluted from the Sephacryl S300 fraction was immunopurified using an anti-
N-CoR affinity column or an irrelevant antibody column as a control. The purified proteins were
separated by SDS-PAGE and immunoblotted using affinity purified anti-N-CoR antibody. FT;
flowthrough fraction. (B) Silver stained SDS-PAGE gel of the affinity purified N-CoR-
1complex. The Sephacryl S300 fractions containing N-CoR were pooled and purified by affinity
chromatography using an anti-N-CoR affinity column as indicated in “Materials and Methods”.
Bound proteins were eluted from the affinity column using 100 mM glycine (pH 2.8) and
separated by SDS-PAGE. Approximate molecular weights of the isolated polypeptides are
indicated on the right. Molecular weight standards are indicated on the left. Control, aliquot from
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the mock affinity purification; N-CoR-1, aliquot from the fraction eluted from the affinity
column. (C) Western blot analysis of the affinity purified N-CoR-1 complex using specific
antibodies. Purified N-CoR-1 was separated by SDS-PAGE, transferred to nitrocellulose and
then probed with the specific antibodies indicated on the left. Control, aliquot from the mock
affinity purification; N-CoR, aliquot from the fraction eluted from the affinity column. (D)
Deacetylase activity of the immunopurified N-CoR-1 complex. Immunopurified N-CoR-1 was
preincubated either alone or with 100 nM Trichostatin A (TSA) and [3H]-core histones. The
reaction was allowed to proceed for 90 min prior to extraction and quantitation of the released
[3H]-acetyl CoA. The results are a representative experiment from at least two independent
purifications.
Figure 3. A representative MS spectrum that identifies the 170 kDa band found in the N-CoR-1
complex as BAF 170. The 170 kDa band was excised using a clean scalpel blade and digested
with trypsin overnight. The peptides were extracted and purified using a ZipTip column prior to
analysis by MALDI-TOF MS as described in “Materials and Methods”.
Figure 4. Components of the SWI/SNF complex copurify with N-CoR-1. (A) Gel filtration
chromatography of the N-CoR-1 associated proteins. The DEAE Sepharose fractions containing
N-CoR-1 were pooled, concentrated and passed through an S300 gel filtration column. Fractions
were collected and aliquots were separated by SDS-PAGE, transferred to nitrocellulose and then
probed with the specific antibodies indicated on the left. A fraction of the proteins identified as
components of the SWI/SNF complex co-elute with N-CoR and migrate as a large molecular
weight complex of approximately 2 MDa. (B) Western blot analysis of the affinity purified N-
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CoR-1 associated proteins. The pooled Sephacryl S300 fractions containing N-CoR were pooled
and purified by affinity chromatography using an anti-N-CoR affinity column as indicated in
“Materials and Methods”. Bound proteins were eluted from the affinity column using 100 mM
glycine (pH 2.8) and were separated by SDS-PAGE, transferred to nitrocellulose and then
probed with the specific antibodies indicated on the left. Input, containing an aliquot of the
pooled Sephacryl S300 fractions; Control, aliquot from the mock affinity purification; N-CoR-1,
aliquot from the fraction eluted from the anti−N-CoR antibody affinity column.
Figure 5. Colocalization of N-CoR and KAP-1. (A) KAP-1 and N-CoR copurify by gel filtration
chromatography. The DEAE Sepharose fractions containing N-CoR-1 were pooled, concentrated
and passed through an S300 gel filtration column. Fractions were collected and aliquots were
separated by SDS-PAGE, transferred to nitrocellulose and then probed with either N-CoR or
KAP-1 antibody as indicated on the left. A major fraction of KAP-1 and N-CoR coelute as a
large molecular weight complex of approximately 2 MDa. (B) KAP-1 is found in the affinity
purified N-CoR-1 complex. The pooled Sephacryl S300 fractions containing N-CoR were pooled
and purified by affinity chromatography using an anti-N-CoR affinity column as indicated in
“Materials and Methods”. Bound proteins were eluted from the affinity column using 100 mM
glycine (pH 2.8), separated by SDS-PAGE, transferred to nitrocellulose and probed with the
specific antibody indicated on the left. Control, aliquot from the mock affinity purification; N-
CoR-1, aliquot from the fraction eluted from the anti−N-CoR antibody affinity column. (C) A
component of N-CoR and KAP-1 colocalize in vivo. Indirect immunofluorescence of HeLa cells
stained with anti-N-CoR and anti-KAP-1 antibodies. Differential interference contrast images
were derived from a single population of HeLa cells grown asynchronously (panel 1) and stained
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Purification of corepressor complexes
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with anti-N-CoR polyclonal antibody (panel 2), or anti-KAP-1 monoclonal antibody (panel 3). In
most cells a significant overlap was observed between KAP-1 and N-CoR staining (panel 4).
Figure 6. N-CoR-2 contains distinct subunits. The P11 500 mM KCl fraction (N-CoR-2) was
subjected to further purification by DEAE Sepharose, gel filtration and affinity chromatography
as described in “Materials and Methods”. (A) Silver stained SDS-PAGE gel of the N-CoR-2
complex. Approximate molecular weights of the isolated polypeptides are indicated on the right.
Molecular weight standards are indicated on the left. (B) Western blot analysis of the affinity
purified N-CoR-2 complex using specific antibodies. The specific antibodies used are indicated
on the left. Control, aliquot from the mock affinity purification; N-CoR-2, aliquot from the
fraction eluted from the affinity column. (C) Deacetylase activity of the immunopurified N-CoR-
2 complex. Immunopurified N-CoR-1 was preincubated either alone or with 100 nM Trichostatin
A (TSA) and in vivo [3H] labelled core histones. The reaction was allowed to proceed for 90 min
prior to extraction and quantitation of the released [3H]-acetylCoA. The results are a
representative experiment from at least two independent purifications.
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Caroline Underhill, Majdi S. Qutob, Siu-Pok Yee and Joseph Torchiaand the corepressor KAP-1
A novel N-CoR complex contains components of the mammalian SWI/SNF complex
published online September 29, 2000J. Biol. Chem.
10.1074/jbc.M007864200Access the most updated version of this article at doi:
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